The overall goal of the project is to achieve holistic understanding of structure and function of heteronuclear metalloenzymes involved in multi-electron redox processes that are more difficult to study than homonuclear enzymes, and to address important scientific issues in the fields of respiration and global nitrogen and sulfur cycles. Specifically, we seek to investigate why a protein containing a heme-non-heme iron center (as in nitric oxide reductase (NOR)) is effective at 2e- reduction of NO, allowing N- N bond formation, whereas a protein containing a heme-copper center (as in heme- copper oxidase (HCO)) is proficient at 4e- reduction of O2, enabling O-O bond cleavage, yet a protein containing a heme-Fe4S4 center (as in assimilatory sulfite and nitrite reductases (SiR and NiR)) is efficient at 6e- reduction of sulfite or nitrite, promoting S-O or N-O bond cleavage, respectively. To achieve this goal, the proposal seeks to overcome a critical methodological barrier to progress in the field by developing a novel biosynthetic approach that uses stable, easy-to-produce, and well-characterized heme proteins as scaffolds for making structural and functional models of HCO, NOR, SiR and NiR, and to use the biosynthetic models to 1) elucidate the roles of heme redox potential (E), electron transfer (ET) rates, and hydrogen bonding (H-bonding) in efficient HCO and NOR activity; 2) investigate structural features responsible for fine-tuning the specific and cross reactivities between HCO and NOR; 3) expand the project scope by designing novel biosynthetic models of the heme-Fe4S4 center in SiRs and NiRs. The models will be fully characterized by crystallography and spectroscopic techniques. Achieving the above goals will result in deeper understanding of the structure and function of HCO, NOR, SiR and NiR that may be very difficult to achieve by studying the native enzymes alone, including the roles of heme E, ET rates, H-bonding, and the identity of nonheme metal ions in HCO and NOR functions, as well as the roles of tetrapyrrole saturation and deformation, and non-covalent interactions (e.g., H-bonding) in SiR/NiR functions. Being able to place different heteronuclear metal centers into the same protein offers insight into similarities and differences between the three heteronuclear metalloenzymes. In doing so, the project will advance the knowledge of metalloprotein structure, function and design in general, as the guiding principles obtained from the studies will be applicable to a broad range of metalloenzymes important for human health in broad terms.